U.S. patent application number 11/659952 was filed with the patent office on 2007-11-29 for micro channel array.
This patent application is currently assigned to Yuji Kikuchi. Invention is credited to Motohiro Fukuda, Yuji Kikuchi, Mitsutoshi Nakajima, Taiji Nishi.
Application Number | 20070276972 11/659952 |
Document ID | / |
Family ID | 35839290 |
Filed Date | 2007-11-29 |
United States Patent
Application |
20070276972 |
Kind Code |
A1 |
Kikuchi; Yuji ; et
al. |
November 29, 2007 |
Micro channel array
Abstract
A microchannel array according to the present invention includes
a first substrate 1, and a second substrate 11 bonded to the first
substrate 1. Two sets, that is, a set of a first through-hole 9, a
first recess 7, and a first groove 8 and a set of a second
through-hole 4, a second recess 2, and a second groove 3 are formed
on the first substrate 1. The different sets are separated by a
partition 5. A cell differentation/proliferation speed after
micro-injection can be increased by using such microchannel
array.
Inventors: |
Kikuchi; Yuji; (Tokyo,
JP) ; Nakajima; Mitsutoshi; (Ibaraki, JP) ;
Nishi; Taiji; (Tokyo, JP) ; Fukuda; Motohiro;
(Ibaraki, JP) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
Kikuchi; Yuji
8-1-1-1902, Minamisenju Arakawa-ku
Tokyo
JP
116-0003
National Agriculture & Food Research Organization
3-1-1, Kannondai Tsukuba-shi
Ibaraki
JP
305-8517
Kuraray Co., Ltd.
1621, Sakazu Kurashiki-shi
Okayama
JP
710-8622
|
Family ID: |
35839290 |
Appl. No.: |
11/659952 |
Filed: |
August 4, 2005 |
PCT Filed: |
August 4, 2005 |
PCT NO: |
PCT/JP05/14304 |
371 Date: |
February 12, 2007 |
Current U.S.
Class: |
710/104 |
Current CPC
Class: |
B01L 3/502761 20130101;
C12M 23/16 20130101; B01F 13/0059 20130101; B01F 5/0453 20130101;
B01F 3/0807 20130101; B01F 5/0456 20130101; B01L 3/502707 20130101;
Y10T 436/25375 20150115; B01L 2200/0668 20130101; B01L 2300/0816
20130101 |
Class at
Publication: |
710/104 |
International
Class: |
G06F 13/00 20060101
G06F013/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 12, 2004 |
JP |
2004-235134 |
Claims
1. A microchannel array containing a first substrate, and a second
substrate bonded to the first substrate with a particle trap
opening edge being formed in an end surface of the first substrate
by a groove formed in a bond surface of the first substrate,
comprising: (a) a first through-hole passed through the first
substrate, a first recess including the first through-hole, and a
plurality of first grooves communicating with the first recess and
the end surface of the first substrate; (b) a second through-hole
passed through the first substrate, a second recess having the
second through-hole, and a second groove communicating with the
second recess and the end surface of the first substrate; and (c) a
partition separating the first recess from the second recess, at
the bond surface of the first substrate, wherein the second groove
is formed between the plurality of adjacent first grooves with the
partition between the second groove and the first grooves.
2. The microchannel array according to claim 1, wherein a width and
depth of the first groove is 0.1 to 300 .mu.m, and a ratio between
the width and depth of the first groove is 1:10 to 10:1.
3. The microchannel array according to claim 1, wherein a width and
depth of the second groove is 0.1 to 300 .mu.m, and a ratio between
the width and depth of the second groove is 1:10 to 10:1.
4. The microchannel array according to claim 1, wherein the
plurality of adjacent first grooves, the second groove formed
between the first groups with the partition between the first group
and the second group, and the second recess corresponding to the
second groove constitute a microchannel group, and a plurality of
microchannel groups are arranged along an end surface of the first
substrate.
5. The microchannel array according to claim 4, wherein the second
through-holes in the second recesses of the plurality of
microchannel groups are connected on a rear side of the first
substrate.
6. The microchannel array according to claim 1, wherein a contact
angle of the surface to water is 0.5.degree. or more and 70.degree.
or less.
7. The microchannel array according to claim 1, wherein at least
one of the first substrate and the second substrate is a
transparent substrate.
8. The microchannel array according to claim 1, further comprising:
at least one additional first substrate bonded to the first
substrate, wherein a plurality of particle trap opening edges
extend in a direction vertical to the bond surface, at end surfaces
of the plurality of first substrates.
9. The microchannel array according to claim 8, the second
through-hole in the additional first substrate is larger than the
second through-hole in the first substrate.
10. The microchannel array according to claim 1, wherein a fluid is
supplied/extracted to/from the end surface of the first substrate
through at least one of the first groove and the second groove of
the microchannel array.
11. A method of supplying/extracting a particle to/from an end
surface of a first substrate through at least one of a first groove
and a second groove of the microchannel array according to claim
1.
12. A method of sucking a particle from at least one of a first
through-hole and a second through-hole in the microchannel array
according to claim 1.
13. A method of manufacturing an oil-in-water type monodispersed
fine particle or water-in-oil type monodispersed fine particle,
comprising introducing a dispersion phase from a second phase of
the microchannel array according to claim 1.
14. A method of manufacturing a core shell type multilayer fine
particle, comprising: introducing a dispersion phase as an internal
particle from a second groove of the microchannel array according
to claim 1 and introducing another dispersion phase as an external
particle from a first groove.
Description
TECHNICAL FIELD
[0001] The present invention relates to a microchannel array, and
more particularly to a microchannel array used for a suction trap
of cells or other such particles.
BACKGROUND ART
[0002] A technique of transferring genes to a cell as typified by
micro-injection is the key to improving bleeding efficiency of
animal and plant species and microbial species having useful
characteristics. Thus, various transferring methods have been
developed hitherto from the viewpoints of reliability and
efficiency.
[0003] For example, the inventors of the present invention
developed such technique that a microcapillary array where a number
of injection probes are regularly arrayed, and a microchamber array
having chambers corresponding to the probes are used to
collectively inject DNAs to all cells held in the micro chamber
array for the purpose of enhancing operation (see Patent Document
1). However, according to this method, it is difficult to
sufficiently cover variations in shape, size, and elasticity
between cells. In some cases, the injection probe cannot be
inserted to a cell, or if inserted, the insertion depth is too
small or large, with the result that DNAs cannot be collectively
injected. Further, a probe insertion direction and a direction in
which an observer observes a target portion with a microscope are
substantially the same. This makes it difficult to insert a probe
while observing a target portion with a microscope in practice.
Even if the target portion can be observed with a microscope, a
probe moves in a focal depth direction of the microscope, leading
to a problem that an image is blurred due to the movement and its
positional control is difficult.
[0004] To that end, the inventors of the present invention have
made extensive studies in view of operational reliability and
developed a microchannel array capable of suction trap of cells or
other such particles at an opening edge of a substrate end surface
(see Patent Document 2). If the microchannel array is used, during
micro-injection, the probe movement direction and the observation
direction of the microscope are substantially orthogonal to each
other, so the injection can be carried out while observing with the
microscope. Thus, DNA or other such substances can be more reliably
and readily injected.
[0005] However, the above microchannel array has a possibility that
cells are damaged upon suction trap of the cells. Further, although
the microchannel array is suitable for micro-injection, the array
is inappropriate for incubation of cells prior to the injection or
differentiation/proliferation of cells after the injection. Thus,
it is necessary to culture cells in another place prior to the
injection, and to carry out differentiation/proliferation of cells
in another place after the injection, resulting in a problem that
the total efficiency of the operation is low. Another serious
problem is that a cell differentiation/proliferation speed cannot
be increased. [0006] [Patent Document 1] Japanese Unexamined Patent
Application Publication No. 2000-23657 [0007] [Patent Document 2]
Japanese Unexamined Patent Application Publication No.
2002-27969
DISCLOSURE OF THE INVENTION
[0007] Problems to be Solved by the Invention
[0008] As described above, the conventional microchannel array has
a problem that a cell differentiation/proliferation speed cannot be
increased. The present invention has been accomplished in view of
the above problems, and an object of the invention is to provide a
microchannel array capable of increasing a cell
differentiation/proliferation speed.
Means for Solving the Problems
[0009] The above object is attained by a microchannel array
containing a first substrate, and a second substrate bonded to the
first substrate with a particle trap opening edge being formed in
an end surface of the first substrate by a groove formed in a bond
surface of the first substrate, including: (a) a first through-hole
passed through the first substrate, a first recess including the
first through-hole, and a plurality of first grooves communicating
with the first recess and the end surface of the first substrate;
(b)a second through-hole passed through the first substrate, a
second recess having the second through-hole, and a second groove
communicating with the second recess and the end surface of the
first substrate; and (c) a partition separating the first recess
from the second recess, at the bond surface of the first substrate,
the second groove being formed between the plurality of adjacent
first grooves with the partition between the second groove and the
first grooves.
Advantages of the Invention
[0010] According to the present invention, it is possible to
provide a microchannel array capable of increasing a cell
differentiation/proliferation speed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows the structure of a microchannel array A
according to the present invention;
[0012] FIG. 2 is a side sectional view showing how the microchannel
array A of the present invention sucks a cell;
[0013] FIG. 3 shows the structure of a rear side of the
microchannel array A of the present invention;
[0014] FIG. 4 is a scanning electron microscope (SEM) photograph of
an outer appearance of the microchannel array A of the present
invention;
[0015] FIG. 5 is a SEM photograph of a microstructure of the center
of the microchannel array A of the present invention;
[0016] FIG. 6 is a SEM photograph of a microstructure of an end
portion of the microchannel array A of the present invention;
[0017] FIG. 7 shows a structure of array C of the present
invention;
[0018] FIG. 8 is a microscope photograph of how a substance is
injected through micro-injection using the microchannel array A of
the present invention;
[0019] FIG. 9 shows a structure of a conventional micro chamber
array; and
[0020] FIG. 10 shows a structure of a conventional micro chamber
array.
DESCRIPTION OF REFERENCE NUMERALS
[0021] 1 FIRST SUBSTRATE [0022] 2 SECOND RECESS [0023] 3 SECOND
GROOVE [0024] 4 SECOND THROUGH-HOLE [0025] 5 PARTITION [0026] 6
BOUNDARY BLOCK [0027] 7 FIRST RECESS [0028] 8 FIRST GROOVE [0029] 9
FIRST THROUGH-HOLE [0030] 10 END SURFACE [0031] 11 SECOND SUBSTRATE
[0032] 15 THIRD GROOVE [0033] 20 CELL
BEST MODES FOR CARRYING OUT THE INVENTION
[0034] Hereinafter, embodiments of the present invention will be
described with reference to the accompanying drawings. The
following description aims at illustrating preferred embodiments of
the present invention and is not construed as limiting the scope of
the present invention to the embodiments. In the following
description, substantially the same components are denoted by like
reference numerals.
[0035] In a microchannel array according to the present invention,
cells or other such particles are suction-trapped at an edge of a
particle trap opening formed in a first substrate end surface. As a
result, upon micro-injection, a micropipette movement direction
(substantially horizontal direction) and an observation direction
of a microscope (vertical direction) are substantially orthogonal
to each other to thereby enable observation with high magnification
and control of a leading edge of the micropipette within a focal
depth. Both of transmitted illumination and reflected
epi-illumination can be used for the observation with a microscope.
The cell or other such particles can be observed with transmitted
illumination and in addition, a microchannel side where recesses or
grooves are formed can be observed with the reflected
epi-illumination. Therefore, DNA or other such substances can be
more reliably and readily injected.
[0036] Further, first grooves are formed on both sides of a second
groove for trapping cells or other such particles with a partition
therebetween. For example, a fresh culture solution can be
continuously supplied to cells or other such particles trapped to
the second groove through a first through-hole and a first recess
from the first groove. Hence, after the cells or other such
particles are suction-trapped in the culture solution, and
micro-injection is carried out, the cells or other such particles
can be cultured as they are in the culture solution, so a series of
operations can be executed with much higher efficiency.
[0037] Referring to FIG. 1, the structure of the first substrate is
described next. FIG. 1 shows the structure of a microchannel array
of the present invention. An upper portion of FIG. 1 is a top view
of the first substrate 1 used in the microchannel array of the
present invention. A lower portion of FIG. 1 is a side sectional
view of how the first substrate 1 and a second substrate 11 are
bonded into a microchannel array. FIG. 1 shows all dimensions in
.mu.m. A first through-hole 9 for supplying a cell culture solution
is formed at the center of the first substrate 1. The first
through-hole 9 is formed in a first recess 7. Then, a first groove
8 extends from the first recess 7 to an end surface 10 of the first
substrate 1. Thus, the first through-hole 9, the first recess 7,
and the first groove 8 communicate with one another. A culture
solution is supplied from the rear side of the first through-hole 9
to thereby flow into the first groove 8 through the first recess 7.
The first groove 8 extends up to the end surface 10 of the first
substrate 1, so the culture solution flows out from the end surface
10. That is, the first groove 8 functions as a microchannel for
supplying a culture solution. As for the first groove 8, two
grooves are paired, and plural first grooves are formed. The first
grooves 8 are arranged at regular intervals, and an interval
between adjacent groove pairs is also constant.
[0038] A substantially U-shaped partition 5 is formed in an area
surrounded by the two first grooves 8 and the first recess 7. A
second groove 3 for sucking cells is provided between the two first
grooves 8 with the partition 5 therebetween. The second groove 3
functions as a microchannel for sucking cells or other such
particles. On the other hand, a second through-hole 4 used for
suction is formed in the partition 5. The second through-hole 4 is
formed in the second recess 2. That is, the second through-hole 4,
the second recess 2, and the second groove 3 communicate with one
another. Cells or other such particles are sucked from the rear
side of the second through-hole 4 and thereby sucked to the edge of
the second groove 3. A boundary block 6 regulating the width of the
first groove 8 is provided between the first grooves 8 of the
adjacent pairs. That is, the first groove 8 width corresponds to a
distance between the partition 5 and the boundary block 6.
[0039] The pair of first grooves 8, the second groove 3 formed
between the pair of first grooves 8 and the second recess 2
corresponding to the second groove 3 form one microchannel group,
and plural microchannel groups are arranged along the end surface
10 of the first substrate 1. That is, in one microchannel group,
three grooves constituting the microchannel are formed. Then, the
second groove 3 is formed between the pair of first grooves 8.
Accordingly, one microchannel group realizes one coaxial
microchannel structure. That is, the first grooves 8 are formed on
both sides of the second groove 3 to attain the coaxial structure.
The structure is repeatedly formed on the bond surface of the first
substrate 1, and thus a coaxial microchannel array having a
plurality of pairs of two microchannels is obtained. The pair of
first grooves 8 may be arranged asymmetrically to the second groove
3 of the microchannel array, or the number of grooves on one side
may be larger than that on the other side.
[0040] Here, the edge of the partition 5 is within the end surface
10. Thus, the first groove 8 and the second groove 3 communicate
with each other around the end surface 10. The edge of the boundary
block 6 reaches the end surface 10 and partitions the adjacent pair
of first grooves 8.
[0041] The first substrate 1 and the second substrate 11 are bonded
with their bond surfaces opposite to each other. In general, the
substrates are closely bonded such that the end surface of the
second substrate 11 is substantially aligned with the end surface
10 of the first substrate. The second substrate 11 is, for example,
a transparent glass substrate or acrylic resin substrate. This
facilitates observation with a microscope. In such state that the
first substrate 1 and the second substrate 11 come into close
contact, there is an opening between the adjacent boundary blocks
6. Then, cells or other such particles are trapped by being sucked
from the second through-hole 4. That is, a particle trap opening
edge is positioned at the second groove 3. The edge of the
partition 5 is within the end surface 10, so a space for cell
differentation/proliferation is defined around the end surface 10.
The space is a recess formed in the end surface 10 of the first
substrate 1, and its width is defined by an interval between
adjacent boundary blocks 6. Further, the space depth is determined
based on a distance from the partition 5 to the end surface 10, and
its height is determined based on a depth of an area where the
first groove 8 and the second groove 3 communicate with each
other.
[0042] The height of the space may be equal to or a little smaller
or larger than a size of a cell or other such particles as a
suction-trap target. The cell is trapped to a narrow space to
thereby grow a cell within a short culture period.
[0043] FIG. 2 shows how a cell 20 is trapped to the end surface 10.
In such state that the first substrate 1 and the second substrate
11 come into close contact, an opening is formed in the end surface
10 in an area corresponding to the second groove 3. Here, if sucked
from the rear side of the second through-hole 4, the cell 20 is
trapped to the opening edge and held. That is, the cell 20 is
partially caught into the space for cell
differentation/proliferation. However, the width of the second
groove 3 is somewhat smaller than that of the cell 20, so the cell
20 is trapped at the edge of the partition 5 as the edge of the
second groove 3. Further, the cell 20 partially extends from the
end surface 10 of the first substrate 1. Thus, the cell can be
easily observed with a microscope, and in addition, genes or other
such substances can be correctly injected through micro-injection.
At this time, the cell 20 partially occupies an area surrounded by
the partitions 5 on both sides thereof, the first substrate 1, and
the second substrate 11, so a culture solution from the first
groove 8 does not flow into the second groove 3 and not weaken an
effect of sucking the cell from the second through-hole 4.
[0044] In general, it is known that a cell
differentation/proliferation speed varies depending on a size of
the space for differentation/proliferation in a cell
incubation/organization test. Therefore, it is preferred that the
space for trapping the cell have substantially the same size as
that of a suction-trap-target cell. Hence, the cell
differentation/proliferation speed can be increased.
[0045] Preferably, the edge of the partition 5 is within the end
surface 10 of the first substrate. On the other hand, it is
preferred that the edge of the boundary block 6 be positioned
closer to the end surface 10 side than the edge of the partition 5.
For example, in the structure of FIG. 1, the width between the
partitions 5 is 80 .mu.m. Thus, a cell having a size of, for
example, about 100 .mu.m is caught in the partitions 5 on both
sides thereof and suction-trapped to the second groove 3. After
that, if the cell is stabilized and its size exceeds that of the
second groove 3, the cell is sandwiched between the first substrate
1 and the second substrate 11. If the cell becomes larger, the
boundary block 6 serves as a guide to immobilize the cells. In this
state, even if the suction force of the suction trap is reduced,
the cell position is not changed, and a cell coat or the like can
be prevented from being damaged due to the suction trap. Moreover,
a cell differentation/proliferation speed can increase.
[0046] The height of the space for cell
stabilization/differentation/proliferation is determined based on
the depth of the first groove 8 and the second groove 3, the width
of the space is determined based on an interval between adjacent
boundary blocks 6, and the depth of the space is determined based
on a distance from the end surface 10 to the edge of the partition
5. In general, the height, width, and depth of the space are
preferably within a range of 0.1 to 300 .mu.m, more preferably 1 to
200 .mu.m.
[0047] The width and depth of the first groove 8 and the second
groove 3 of the microchannel are preferably within a range of 0.1
to 300 .mu.m, more preferably 1 to 200 .mu.m. Further, a ratio
between the width and depth of the groove is preferably selected
within a range of 1:10 to 10:1 as appropriate based on a shape or
deformability of a target cell or other such particles.
[0048] If the first substrate 1 and the second substrate 11 are
brought into close contact when in use, impurities such as a cell
fragment coexist with a cell sample. Thus, there is a fear that the
impurities are sucked to clog a suction port. To prevent this
clogging, both substrates may be laminated and brought into close
contact instead of completely bonding these substrates through the
application of ultrasonic wave, laser, or heat. If so, the
substrates can be separated and washed after used, clogged
impurities can be readily removed, and the substrates can be
reused.
[0049] The microchannel array of the present invention is
structured such that grooves of the bond surface of the first
substrate are arranged in parallel along the end surface 10. Owing
to such structure, micro-injection efficiency can be more enhanced.
That is, even when some suction ports are clogged with impurities
such as a cell fragment, if cells or other such particles are
suction-trapped to other ports, the micro-injection can be
continued.
[0050] In the microchannel array of the present invention, as in
the structure of FIG. 1, first recesses 7 are formed and connected
to the plural first grooves 8 arranged in parallel, with the result
that the first grooves 8 communicate with each other. The first
recess 7 has a first through-hole 9. It is possible to form plural
first through-holes 9, but the number of first through-holes 9 is
preferably small from the viewpoint of manufacturing costs of a
microchannel array. Further, in the case of supplying a culture
solution or the like from the first through-hole 9, it is necessary
to execute positional alignment between a culture solution supply
port and the first through-hole 9. Thus, it is preferred that the
number of first through-holes 9 be small from this point of view as
well. Preferably, only one first through-hole 9 is formed. At this
time, the first through-hole 9 preferably has a size enough to
supply a sufficient amount of culture solution to each channel.
[0051] On the other hand, the second through-hole 4 is generally
formed in every second recess 2 surrounded by the partition 5. In
this case, plural second through-holes 4 are formed. FIG. 3 is a
plan view showing the structure of the rear side of the first
substrate 1 (the side opposite to the bond surface). As shown in
FIG. 3, the first through-hole 9 and the second through-hole 4 are
formed on the rear side of the first substrate 1. Plural second
through-holes 4 as many as the second grooves 3 are formed. The
adjacent second through-holes 4 communicate with each other through
the third groove 15. Aside from this structure, for example, the
second through-hole 4 may be formed in a third recess, and plural
second through-holes 4 may be connected. As a result, the suction
force is uniformly applied to the plural second through-holes 4,
and a cell coat or the like can be prevented from being damaged due
to excessive suction. Further, it is necessary to execute
positional alignment between the suction port and the second
through-holes 4 upon the suction. From this point of view as well,
it is preferable that the second through-holes be connected
together.
[0052] The diameter and width of the second through-hole 4 are
preferably selected as appropriate within a range of 10 to 2,000
.mu.m, more preferably, 20 to 1,000 .mu.m in accordance with the
number of microchannels and a requisite suction force level.
[0053] Examples of a method of manufacturing the microchannel array
of the present invention include resin molding with a metal
structure, precision machining, wet etching, dry etching, laser
processing, and electric-discharge machining. An appropriate method
is preferably selected from these manufacturing methods based on an
application, requisite accuracy of finishing, and costs.
[0054] A method of manufacturing a resin mold through resin molding
with the metal structure used as a mold enables copying the shape
of the metal structure to the resin mold with high accuracy and
excels in high dimensional accuracy. Further, a general-purposed
resin material can be used to lower a material cost, so this method
is appropriate for mass production. The above metal structure may
be used as a metal mold as it is or may be set in a prepared metal
mold when in use.
[0055] Examples of a manufacturing method of the metal structure
include plating to a resist pattern formed through
photolithography, precision machining, wet etching, dry etching,
laser processing, and electric-discharge machining. Preferably, an
appropriate method is selected based on an application, a requisite
accuracy of finishing, a cost, or the like.
[0056] As a method of manufacturing a resin mold, for example,
there are injection-molding, press-molding, monomer-cast molding,
solvent-cast molding, hot emboss molding, and roll transfer based
on extrusion molding. From the viewpoint of productivity and mold
transferability, injection-molding is preferred. The
injection-molding can produce 10000 to 50000 resin molds, 200000
resin molds under certain circumstances, from one metal structure,
making it possible to considerably reduce a manufacturing cost of
the metal structure. Further, a required period for 1 cycle of the
injection-molding is as short as 5 to 30 seconds, so this method is
very useful in terms of productivity. If multicavity-mold metal
structure is used, the productivity can be more improved.
[0057] There is no particular limitation on a resin material. For
example, an acrylic resin, a polylactic acid, polyglycolic acid, or
styrene-based resin, a methacrylate/styrene copolymer resin (MS
resin), a polycarbonate resin, a polyester resin such as
polyethylene terephthalate, a polyvinyl alcohol resin,
ethylene/vinyl alcohol copolymer resin, a thermoplastic elastomer
such as styrene-based elastomer, a vinyl chloride resin, a silicone
resin such as polydimethyl siloxane, and a polyvinyl butyral resin.
These resins may optionally contain one or two or more of a
lubricant, a light stabilizer, a thermal stabilizer, an
anti-fogging agent, a pigment, a flame retardant, an antistatic
agent, a mold lubricant, an anti-blocking agent, a UV absorber, and
an antioxidant.
[0058] In the case of suction-trapping cells or other such
particles, if the end surface of the first substrate and the second
substrate is rough, there is a possibility that the cell coat is
damaged when in contact with the surface. Thus, it is desirable to
form the end surfaces as smoothly as possible. The method of
smoothing the surface is appropriately selected based on a
substrate material. If the substrate material is silicone, laser
cutting and polishing in two steps with a not-fine dicing cutter
and a fine dicing cutter are executed, for example. If the
substrate material is a resin, fusing with thermal cutter, laser
cutting, dicing, chamfering through machining, polishing, and the
like are carried out.
[0059] A method of forming the first through-hole 9 and the second
through-hole 4 is also selected as appropriate based on a substrate
material. If the substrate material is silicone, laser cutting, wet
etching, dry etching, sand blasting, and the like are carried out.
If the substrate material is a resin, laser cutting, laser
ablation, machining, and the like are carried out. A contact angle
of the surface of the microchannel array of the present invention
to water is preferably 0.5.degree. or more and 70.degree. or less,
more preferably, 1.degree. or more and 50.degree. or less.
[0060] A space defined by the recesses and grooves of the
microchannel array of the present invention can function as a flow
path if having an affinity with an aqueous liquid such as a cell
culture solution, a saline, a blood sample, and a reagent, that is,
hydrophilic nature. If not hydrophilic, the aqueous liquid cannot
flow through a flow path or hardly flows. Further, there is a fear
that bubbles are mixed to hinder the flow of the aqueous
liquid.
[0061] In particular, in the case of using a blood sample, the
space should be hydrophilic. Blood cells in a blood component
(erythrocyte, leucocyte, and blood platelet) tend to adhere to a
hydrophobic surface. Thus, if the flow path surface is hydrophobic,
the cells adhere to the flow path and clog the flow path in some
cases.
[0062] For example, the contact angle of polymethylmethacrylate
generally used as a substrate material to water is about
68.degree., the contact angle of a polycarbonate resin is about
70.degree., and the contact angle of a polystyrene resin is
84.degree.. Under certain circumstances, an affinity imparting
process should be executed to reduce the contact angle.
[0063] As a method of making a material surface hydrophilic, there
are two broad categories: chemical treatment and physical
treatment. Examples of the chemical treatment include chemical
treatment, solvent treatment, coupling agent treatment, monomer
coating, polymer coating, inorganic material coating, steam
treatment, surface grafting, electrochemical treatment, and anodic
oxidation. Examples of the physical treatment include UV
application, plasma contact treatment, plasma jet treatment, plasma
polymerization, vapor deposition polymerization, thermal oxidation,
ion beam application, and mechanical treatment. Applicable
treatment methods are described below.
[0064] Coating is a method of coating a material surface with a
hydrophilic polymer (polyvinyl alcohol or the like) in an aqueous
solution through, for example, dipping, spin-coating, or the like,
and then drying the surface. If the microchannel array is too
hydrophobic, there is a fear that a coating material is repelled by
the surface, a uniform coating film thickness cannot be attained,
and a reforming effect fluctuates. In some cases, a coating
material needs to be selected. A material applicable to a
hydrophobic surface is, for example, "Lipidure-PMB" (Lipidure-PMB)
(copolymer of MPC polymer having a phospholipid polar group and
butylacrylate) available from NOF Corporation. This method requires
no large device and attains a beneficial effect through simpler
steps, but has a possibility that coating is peeled off during
ultrasonic washing or the like. In this case, it is preferred to
coat the surface again or dispose of the cell before reuse.
[0065] Vacuum evaporation is a kind of steaming. This method heats
a substance in a vacuum (at a pressure of 10.sup.-2 Pa or less) and
vaporizes the substance to apply the steam to a desired material
surface. This method does not require a large device and can
process the target with the lower degree of vacuum. Thus, this
method is advantageous in terms of costs.
[0066] Sputtering is a kind of plasma treatment. This method
accelerates cations resulting from low-pressure glow discharge in
an electric field to cause the cations to collide against a cathode
to thereby spatter substances on the cathode side to cause the
substances to deposit on the anode. This method can adopt a variety
of materials. For example, an inorganic material such as SiO.sub.2
or Si.sub.3N.sub.4 can be used. Further, the thus-completed product
withstands ultrasonic washing and thus can be repeatedly used.
Further, it excels in anti-solvent property, so an elute does not
damage the cells or the like. This method is thus applicable to the
bioengineering field.
[0067] Further, sputtering enables uniform deposit film thickness.
For example, if an inorganic material such as SiO.sub.2 or
Si.sub.3N.sub.4 is deposited to 10 nm to 300 nm to thereby attain a
hydrophilic material surface. Further, if SiO.sub.2 is deposited to
10 nm to 50 nm, a transparent and hydrophilic surface can be
obtained.
[0068] To bring the material surface and the deposit film into
close contact through sputtering, it is necessary to remove water
from the material beforehand. In addition, it is effective to etch
the material surface with argon gas or the like or predeposit an
inorganic material such as chromium having high adherence.
[0069] In the case of executing sputtering, requisite
heat-resistance temperature is about 50.degree. C. to 110.degree.
C. Thus, if a thermoplastic resin is used as a material, it is
necessary to meet conditions of (1) a thermoplastic resin having a
glass transition temperature not less than the above temperature,
for example, polycarbonate is selected, and (2) a sputtering period
is shortened (film thickness is reduced).
[0070] Implantation is a kind of plasma treatment. This method
activates, if a thermoplastic resin is used as a material,
molecules of the material surface with plasma to recombine
generated radicals to implant new functional groups into the
material surface. According to this method, the material surface
can be imparted with hydrophilic property and other properties.
[0071] Plasma polymerization is a kind of plasma treatment. This
method vaporizes a monomer as a material of a polymer to
vapor-phase transfer the vaporized one and activates the monomer
through electron collision excitation in plasma to cause
polymerization to form a polymer film on the material surface. This
method is advantageous in that a film thickness can be easily
controlled and unreacted monomer or solvent does not remain. Thus,
the cell is not damaged with the unreacted monomer or solvent, and
this method is applicable to the bioengineering field.
[0072] Vapor deposition polymerization is substantially the same as
the above plasma polymerization except that a monomer is activated
with heating to cause polymerization.
[0073] Thermal oxidation is a method of exposing, in the case of
using silicone or other such inorganic materials, the material to
the atmosphere under the high-temperature condition to thereby
oxidize the material surface. In general, the material is exposed
to an oxygen plasma atmosphere in a vacuum device to accelerate
thermal oxidation.
[0074] Excimer UV treatment is a kind of UV irradiation treatment.
The method applies, in the case of using a thermoplastic resin as a
material, UV with a light emission center wavelength of 120 nm to
310 nm by use of an excimer lamp using a discharge gas such as
argon, krypton, and xenon to dissociate molecules on the material
surface to extract light hydrogen atoms to form highly hydrophilic
functional groups such as a hydroxyl group. According to this
method, a requisite heat resistance temperature for making the
thermoplastic resin hydrophilic is low. Thus, this method is
applicable to polymethylmethacrylate having a glass transition
temperature of 100.degree. C.
[0075] According to the excimer UV treatment, although a
hydrophilic property is enhanced as an UV exposure amount
increases, adhesion is imparted to the surface in some cases,
resulting in another problem. Thus, an exposure amount should be
adjusted in accordance with a requisite hydrophilic level.
[0076] As another method of forming a hydrophilic surface, there is
a method of selecting an appropriate material. For example, if a
blood cell is treated, it is necessary to not only immobilize the
blood cell to the hydrophobic surface but also to suppress
deposition of blood platelets to the surface due to blood
coagulation. Examples of preferred materials include a material
containing heparin preventing coagulation of blood platelets, a
material where an enzyme urokinase that dissolves a blood
platelet-derived thrombus is immobilized, a material covered with a
polymer material having high water content such as polyvinyl
alcohol, acrylamide, or polyethylene glycol, which can prevent
deposition of blood platelets or protein to the surface, and a
material covered with a micro phase separation structure that
prevents activation of blood platelet. A final separation size of
the micro phase separation structure is generally 20 nm to 20
.mu.m, and a material of uniform microdomain structure is
preferred. Such structure is realized by, for example, a
combination of amorphous/amorphous, hydrophilic/hydrophobic, or
crystalline/non-crystalline, glass/liquid materials. Specific
examples thereof include a hydroxyethyl methacrylate (HEMA)-styrene
copolymer, a HEMA-butadiene copolymer, a HEMA-styrene block
copolymer, and a blend of crystalline nylon 610 and amorphous
polypropylene oxide.
[0077] In addition, "EXCEVAL" available from Kuraray or polyvinyl
butyral resin is preferred in some cases. In this case, to keep a
fine groove shape, the temperature of the used aqueous solution is
set to 70.degree. C. or less, and an observation target should not
be immersed in the water for long time.
[0078] One or both of the first substrate 1 and the second
substrate 11 of the microchannel array of the present invention are
preferably transparent. As a result, cells or other such particles
and grooves or recesses of the microchannel array can be observed
with a microscope with transmitted illumination or reflected
epi-illumination, and micro-injection efficiency can be enhanced.
An optical property that defines substrate transparency is a total
transmittance of 80% or more and a haze value of 10% or less in the
case of using a 1 mm-thick plate.
[0079] Further, if a high UV transmittance is required upon
observation with a microscope, glass, especially, quartz glass is
preferred as a material. If a thermoplastic resin is used, a
material added no UV absorber or a material the molecular structure
of which does not have an ability to absorb UV such as a ring
system is preferred.
[0080] Regarding an outer size of the microchannel array of the
present invention, the length and with are set to 100 mm or less in
view of manufacturing costs, and an appropriate size is desirably
selected in accordance with applications. The flatness of the
microchannel array is preferably 1 .mu.m or more from the viewpoint
of industrial reproducibility, more preferably 200 .mu.m or less in
view of the microchannel array laminate as described below.
Although not particularly limited, the microchannel array thickness
is preferably 0.2 to 10 mm in view of damage, deformation, and
distortion when in use. A dimensional accuracy of a shaping portion
is within a range of .+-.0.5 to 10% of the thickness from the
viewpoint of industrial reproducibility.
[0081] In the microchannel array of the present invention, plural
first substrates are piled up in close contact while being aligned,
and thus plural trap opening edges are formed in the length
direction. That is, on the rear side of the first substrate 1,
which is the side opposite to the bond surface of the second
substrate 11, another first substrate 1 can be laminated. At this
time, the substrates are laminated such that a surface having a
groove of the additional first substrate 1 faces the second
substrate. Thus, the first grooves 8 are arranged in the length
direction of the first substrate 1, and the trap opening edges
extend in the direction vertical to the bond surface. In this way,
the micro-injection efficiency can be further enhanced by forming
plural end surface 10 structures. The first substrate 1 is not
limited to a two-layer structure, and three or more layers may be
laminated.
[0082] As a positional alignment method of each substrate, there is
a method of forming convex and concave patterns on the front and
rear sides of the substrate and bringing the substrates into close
contact with high positional accuracy, a method of fixing an outer
edge of the substrate with a jig, a method of fixing the substrate
by inserting a positioning pin to a through-hole, or a method of
observing and positioning the substrate with an optical device such
as a CCD camera or laser.
[0083] It is preferred that the additional first substrate 1 have
the second through-hole 4 larger than the second through-hole 4 of
the first substrate 1.
[0084] Along with the development of a high-density microchannel
array, the second through-hole 4 of a smaller diameter is required.
In this case, there is a possibility that upon micro-injection, a
suction force enough to suck cells or other such particles cannot
be obtained. On the first substrate 1; another first substrate
having a second through-hole 4 the diameter of which is larger than
that of the first substrate 1 is layered such that positions of the
through-holes are aligned. Hence, even in the high-density
microchannel array, a suction force necessary to suction-trap cells
or other such particles can be obtained.
[0085] The microchannel array of the present invention uses a
thermoplastic resin as a material and thus can be incinerated as
infectious wastes similar to a thermoplastic resin of a blood
circuit used in blood purification treatment such as artificial
dialysis or plasma exchange. Such microchannel arrays can be burned
up and overcome a problem of increasing an amount of wastes
resulting from a single-use system. In addition, a resin substrate
is laminated, so it is unnecessary to separate garbage according to
type and to collectively burn the substrates. Further, a
thermoplastic resin containing no halogen such as
polymethylmethacrylate is used to prevent generation of dioxin as
hazardous substance. The resin can be readily burned in an
incinerator at ordinary temperature used in general waste
incinerator and recycles as heat resources.
[0086] The microchannel array of the present invention supplies or
extracts fluids such as a cell culture solution, a saline, a blood
sample, or a reagent from an opening edge and thus is promising in
the biotechnology field, the medical field, the agricultural field,
and the industrial field.
[0087] If the microchannel array of the present invention is used
to supply and extract cells or particles, the microchannel array
can be expected to find its application to classification of cell
and blood components, and to reaction and synthesis in the
industrial field.
[0088] In the case where the microchannel array of the present
invention is used to trap cells or particles, the microchannel
array is expected to exert beneficial effects in the biotechnology
field, especially, upon micro-injection.
[0089] The microchannel array of the present invention enables
formation of oil-in-water type monodispersed fine particles or
water-in-oil type monodispersed fine particles of uniform particle
size and is expected to find application in the agricultural field,
the medical field, the foods field, and the industrial field.
[0090] A dispersion phase as a particle is introduced to the end
surface 10 from the second through-hole 4 through the second groove
3. Owing to a difference in wettability between the substrate
surface and the dispersion phase, fine particles of uniform
particular diameter can be formed. In the case of forming the
oil-in-water type monodispersed fine particle, the substrate
surface should be made hydrophilic. On the other hand, in the case
of forming the water-in-oil type monodispersed fine particle, the
surface should be made hydrophobic. Based on the method of
controlling a control angle to the water, an appropriate one is
selected.
[0091] It is possible to manufacture core shell type multilayer
fine particles with the microchannel array of the present
invention. A dispersion phase as an inner particle is introduced to
the end surface 10 from the second through-hole 4 through the
second groove 3. At the same time, a dispersion phase as an
external particle is introduced to the end surface 10 from the
first through-hole 9 through the first groove 8. As a result, core
shell type multilayer fine particles of uniform particle size can
be efficiently manufactured.
EXAMPLE
[0092] Next, the conventional micro chamber array and microchannel
array and the coaxial microchannel array of the present invention
are compared.
Production Example 1
Fabrication of Micro Chamber Array
[0093] 100 through-holes having the diameter of 60 .mu.m were
formed in a 0.2-mm thick silicon substrate with the length of 20 mm
and the width of 20 mm by use of a dry etching device available
from Alcatel. The micro chamber array is used as Comparative
Example. FIG. 9 shows the outer appearance of the micro chamber
array.
Production Example 2
Fabrication of Microchannel Array
[0094] 14 channels having the width of 80 .mu.m, the depth of 100
.mu.m, and the length of 5000 .mu.m were formed in a 0.3-mm thick
silicon substrate with the length of 10 mm and the width of 20 mm
by use of a dry etching device available from Alcatel. The channels
communicated with each other through the grooves, and then a
through-hole having the diameter of 3 mm was formed in the groove
through sand blasting. Then, an acrylic substrate having the same
size as that of the silicon substrate was brought into contact with
the silicon substrate to complete a microchannel array. FIG. 10 is
an outer appearance of the microchannel array.
Production Example 3
Coaxial Microchannel Array A
[0095] First, a resist pattern was formed on a glass plate through
photolithography, and the resist pattern was subjected to plating
to manufacture the metal structure. Next, the metal structure was
used as a mould, and an acrylic resin (PARAPET G-HS) available from
Kuraray was used as a material. A first substrate of a coaxial
microchannel array A was manufactured through the
injection-molding. Further, the through-hole (second through-hole
4) having the diameter of 0.3 mm was formed for sucking a cell on
the substrate with a machining center. Finally, an acrylic
substrate (second substrate) having substantially the same as that
of the acrylic substrate was brought into close contact with the
substrate to complete a coaxial microchannel array A of FIG. 1.
[0096] A shape of the coaxial microchannel array A has 20
microchannel pairs on a substrate having the width of 16 mm, the
length of 8 mm, and the thickness of 1.0 mm. Each microchannel
includes a first groove and a second groove through which a first
through-hole, a second through-hole, a first recess, a second
recess, a first recess, and a substrate end surface communicate
with each other. The second groove 3 for sucking cells or the like
has the width of 80 .mu.m and the depth of 150 .mu.m. The first
grooves 8 for supplying a culture solution on both sides of the
second groove 3 separated by the partition has the width of 100
.mu.m and the depth of 150 .mu.m. A positional relation between the
boundary block 6 between the coaxial microchannels and the edge of
the partition 5 at the edge of the end surface 10 is that the end
surface 10 is at the same position as the edge of the boundary
block 6, and the edge of the partition 5 is positioned 50 .mu.m
behind the edge of the boundary block 6.
[0097] A contact angle measuring apparatus (Kyowa Interface
Science, CA-DT/A type) was used to measure a contact angle of the
coaxial microchannel array A to the water in the air. The
measurement result was 70.degree.. Further, SEM observation of the
coaxial microchannel array A was carried out. FIG. 4 shows the
outer appearance, FIG. 5 shows the center of the fine structure,
and FIG. 6 shows an end portion.
Production Example 4
Fabrication of Coaxial Microchannel Array B
[0098] The same process as that of Production Example 3 was carried
out to fabricate a coaxial microchannel array B except that before
the first substrate and the second substrate were brought into
close contact, SiO.sub.2 was deposited to 100 nm on both substrates
to execute surface modification with a sputtering device (SV
available from ULVAC). A result of measuring a contact angle of the
coaxial microchannel array B to the water in the air was
11.degree..
Production Example 5
Fabrication of Coaxial Microchannel Array C
[0099] First, a glass substrate was subjected to precision
machining with a micro cutting tool of the diameter of 100 .mu.m to
complete the metal structure. Next, the metal structure was used as
a mould, and an acrylic resin (PARAPET G-HS) available from Kuraray
was used as a material. A first substrate of a coaxial microchannel
array C was manufactured through injection-molding. Further, a
through-hole for sucking cells or the like, which has the diameter
of 0.3 mm (second through-hole 4) was formed in the substrate
through a machining center. Further, SiO.sub.2 was deposited to 100
nm onto the first substrate and an acrylic substrate (second
substrate) having substantially the same size as the first
substrate for surface modification with a sputtering device (SV
available from ULVAC). Finally, the two substrates were brought
into close contact to complete a coaxial microchannel array C of
FIG. 7.
[0100] A shape of the coaxial microchannel array C is similar to a
shape of the coaxial microchannel array A of Production Example 3
except that the depth of the second groove 3 for sucking cells or
the like and the depth of the first grooves 8 for supplying a
culture solution on both sides of the second groove 3 separated by
the partition 5 were each 200 .mu.m.
[0101] A result of measuring a contact angle of the coaxial
microchannel array C to the water in the air was 15.degree..
Comparative Example 1
Micro-Injection with Micro Chamber Array
[0102] The micro chamber array manufactured in Production Example 1
was used to hold a bovine ova cell on the surface of the micro
chamber array in a culture solution, followed by micro-injection.
The micro chamber array made of silicone does not transmit visible
light. Thus, the cell was observed with reflected epi-illumination,
but the light was not reflected by a through-hole portion, and the
cell could not be well observed.
[0103] Further, the probe movement direction and the observation
direction of a microscope are substantially the same (vertical
direction), and the probe is moved in the focal depth direction of
the microscope. Thus, an image is blurred due to the movement and
its positional control is difficult.
Comparative Example 2
Micro-Injection with Microchannel Array
[0104] The microchannel array manufactured in Production Example 2
was used to hold a bovine ova cell to the end surface of the
microchannel array in a culture solution to carry out
micro-injection. In a microchannel array that suction-traps cells
or other such particles at an opening edge of the substrate end
surface, the particle trap opening edge can be freely accessed from
the outside to enable observation with a microscope with
transmitted illumination and reflected epi-illumination. Further, a
micropipette movement direction (substantially horizontal
direction) and an observation direction of a microscope (vertical
direction) are substantially orthogonal to each other. Thus, even
when the micropipette was moved, the cell could be observed with
high magnification.
[0105] However, the micro-injection is one of a series of processes
such as suction-trap of cells in a culture solution,
micro-injection, and cell incubation in a culture solution, and its
efficiency is low. A cell differentation/proliferation speed in the
culture solution was the same as that of the related art. An ova
cell having a size of 100 .mu.m was differentiated/proliferated up
to about 200 .mu.m after about 5 days. Further, the following
problem remains to be solved. That is, if suction force necessary
for trap is applied to a cell, the cell coat is damaged upon
suction-trapping cells or other such particles to a particle
opening edge.
Example 1
Micro-Injection with Coaxial Microchannel Array A
[0106] A bovine ova cell was immobilized in a culture solution
containing the bovine ova cell with the coaxial microchannel array
A manufactured in Production Example 3. As a result of observation
with a microscope with transmitted illumination, it was observed
that a bovine ova cell was trapped to an opening of the coaxial
microchannel (second groove 3) of four coaxial microchannel pairs
among 20 coaxial microchannel pairs. Bubbles were partially mixed
in the second groove 3 and first groove 8 but did not influence the
suction and culture solution supply. Since ova cells just after
sampled were used, the cells were stabilized while supplied with
fresh culture solution from the first groove 8 over 2 days.
[0107] Next, micro-injection was carried out through observation
with a microscope with transmitted illumination. The micropipette
movement direction (substantially horizontal direction) and the
observation direction of a microscope (vertical direction) are
substantially orthogonal to each other. Thus, the cell could be
observed with high magnification, and a probe could be injected to
the depth optimum for the cell coat of one ova cell without
damaging the ova cell. FIG. 8 is a microscope photograph of the
cell and coaxial microchannel array A upon micro-injection. The
cell could be sucked from the second groove 3 formed in the lower
first substrate 1. Then, a culture solution could be supplied from
the first grooves 8 on both sides thereof. A substance such as gene
could be injected to the cell with a cell-insertion glass
micropipette.
[0108] Next, as a result of carrying out
differentiation/proliferation of a bovine ova cell in a coaxial
microchannel, it was confirmed that 100-.mu.m ova cell grew to
about 200 .mu.m in third day. Under such environment that a fresh
culture solution is continuously supplied from the first groove 8,
the cell was cultured in a narrow space having the height of 150
.mu.m and the depth of 50 .mu.m, with the result that the cell
differentation/proliferation speed could increase.
[0109] A series of processes, stabilization, micro-injection, and
cell differentation/proliferation in a culture solution can be
carried out by using the coaxial microchannel array, and
micro-injection efficiency can be drastically improved. Further,
the reason the trapped ova cell could be held without damaging its
cell coat over a few days is that the ova cell could be immobilized
with the minimum suction force in a small.
Example 2
Micro-Injection with Coaxial Microchannel Array B
[0110] Similar to Example 1, a series of processes, stabilization,
micro-injection, and cell differentation/proliferation in a culture
solution were executed with the coaxial microchannel array B
fabricated in Production Example 4. As a result, similar to the
coaxial microchannel array A, the micro-injection efficiency could
be drastically improved, and the mixture of bubbles observed in
Example 1 did not occur in this Example, and bubbles could be
completely eliminated through an affinity imparting process.
Example 3
Micro-Injection with Coaxial Microchannel Array C
[0111] The coaxial microchannel array C obtained in Production
Example 5 was used to execute a series of processes, stabilization,
micro-injection, and cell differentation/proliferation in culture
solution, similar to Example 1. Similar to the coaxial microchannel
array B, the micro-injection efficiency could be drastically
enhanced, and bubbles could be completely removed in an affinity
imparting process. On the other hand, 100 .mu.m ova cell grew to
about 200 .mu.m through differentation/proliferation over 4 days
that is 1 day longer than Examples 1 and 2. It is supposedly
because the space height is as large as 200 .mu.m, and an
appropriate space size needs to be selected in accordance with a
target cell size.
INDUSTRIAL APPLICABILITY
[0112] According to the present invention, a microchannel array
capable of increasing a cell differentation/proliferation speed can
be provided.
* * * * *